QC Report

	general
Report generated at	2025-02-21 23:41:53
Title	Pancreas
Description	ATAC-seq data from Liu et al. pancreas data processed in 2025
Pipeline version	v2.2.0
Pipeline type	atac
Genome	mm10
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	35834434	107188668
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	33346717	99968988
Mapped Reads (QC-failed)	0	0
% Mapped Reads	93.10000000000001	93.30000000000001
Paired Reads	35834434	107188668
Paired Reads (QC-failed)	0	0
Read1	17917217	53594334
Read1 (QC-failed)	0	0
Read2	17917217	53594334
Read2 (QC-failed)	0	0
Properly Paired Reads	30425544	92017284
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	84.89999999999999	85.8
With itself	31906314	95763600
With itself (QC-failed)	0	0
Singletons	1440403	4205388
Singletons (QC-failed)	0	0
% Singleton	4.0	3.9
Diff. Chroms	77156	74573
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	12060096	36323994
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	654079	2445759
Paired Optical Duplicate Reads	0	0
% Duplicate Reads	5.4235	6.7332

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	33198628	99465529
Rm = Number of Mitochondrial Reads	184786	618828
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.005535263709098176	0.006183064152572814

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	22760276	67614240
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	22760276	67614240
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	22760276	67614240
Paired Reads (QC-failed)	0	0
Read1	11380138	33807120
Read1 (QC-failed)	0	0
Read2	11380138	33807120
Read2 (QC-failed)	0	0
Properly Paired Reads	22760276	67614240
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	22760276	67614240
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.7115782862880241	0.7100783510400204
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	2.690391927331464	2.6385425585045663
NFR / mono-nuc reads (QC pass)	True	True
NFR / mono-nuc reads (QC reason)	OK	OK
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	False	False

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.5968982977183581	0.578667008606471
Fraction of Reads in blacklist regions	0.006021499915027393	0.003990283703551205
Fraction of Reads in promoter regions	0.1798394711909469	0.17362450572542115
Fraction of Reads in enhancer regions	0.412723202477861	0.4024473394953489

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	12023511	36196537
Distinct Fragments	11390159	33867478
Positions with Two Read	491605	1671907
NRF = Distinct/Total	0.947324	0.935655
PBC1 = OneRead/Distinct	0.95317	0.944815
PBC2 = OneRead/TwoRead	22.084322	19.138931

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	128839	75160
N1	85488	41164
N2	124650	71960
Np	136473	81579
N optimal	136473	81579
N conservative	128839	75160
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0592522450500237	1.0854044704630121
Self Consistency Ratio	1.4580993823694555	1.74812943348557
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	153507	199099

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	191.0	209.0	486.0	343.0
50 percentile (median)	286.0	332.0	745.0	538.0
75 percentile	520.0	626.0	1094.0	876.0
Max size	2444.0	3082.0	3087.0	3087.0
Mean	408.490765893412	479.91451991220447	824.6770124664436	659.4317484044464

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	12.868879168579248	12.728467987530006

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.12161090374617473	0.16077010805100822
Synthetic AUC	0.48996389787314965	0.49418751744417083
X-intercept	0.39046278374098187	0.17221617903476077
Synthetic X-intercept	2.758692794537362e-84	6.740138456396787e-254
Elbow Point	0.7484923135987714	0.7641490569961448
Synthetic Elbow Point	0.49922269426240773	0.5055456531927898
Synthetic JS Distance	0.456881003096886	0.4749334025884419

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.3235066657363909	0.3221814073485112	0.31360674185146087	0.30747324823883254	0.312779335364826	0.30804851167446384	0.3337652065544672	0.315363083106304	0.3151767473919307

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.29460196500526764	0.27287393175724234	0.2886000049693674	0.3018841063558227

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.24649442106002536	0.19988544954375773	0.2413224344457617	0.25640433858589073

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates